ABSTRACT Engineered probiotics represent a powerful tool with which to ?knock-in? gene functions and pathways into the gut microbiome, alter the structure of the gut microbiome to test hypotheses regarding community architecture and disease, and to deliver therapeutics. However, known probiotics fail to persist in the gut due to colonization resistance by the gut microbiota, limiting their value as either research or therapeutic tools. Further, controlled delivery of therapeutics and other gene products by engineered probiotics is limited by a lack of robust and tunable synthetic biology tools for the complex in vivo environment. The rationale for the proposed research is that the promise of probiotic therapies is currently limited by poor persistence and a lack of robust engineering tools. The central motivation for this proposal is to understand the host and microbial mechanisms governing probiotic integration and develop tools to engineer probiotic therapies. Guided by strong preliminary data, this interdisciplinary proposal will pursue three specific aims: to 1) identify determinants of probiotic colonization in the gut, 2) design and optimize gut-relevant biosensor and expression circuits, and 3) demonstrate the efficacy of in vivo delivery of a phenylketonuria (PKU) therapeutic by an enhanced probiotic colonizer. The first aim of this proposal is to optimize and identify mechanisms of probiotic colonization, testing the hypothesis that probiotic gut colonization is enhanced and tunable by modulating expression of colonization factors selected from fecal metagenomes. In particular, we will select for durable colonizers from probiotics expressing an exhaustive combinatorial library of colonization factors driven by in vivo characterized promoters. The second aim is to develop synthetic biology tools for tunable gene expression control and biocontainment of engineered probiotics, testing the hypothesis that combining sensors for temperature, pH, bile acids, and short chain fatty acids will enable spatial control over gene expression along the gastrointestinal tract. The third aim is to demonstrate that the engineered probiotic chassis can reliably deliver therapeutics to the gut, testing the hypothesis that our engineered probiotic can stably deliver phenylalanine-ammonia lyase (Pal2) in a murine model of PKU to decrease serum phenylalanine. This proposal is innovative because our integrated and complementary research team will improve understanding of probiotic therapies at both basic science and translational levels. The proposed experiments are significant in that they will 1) improve our understanding of the genetic elements and microbial interactions governing gastrointestinal colonization, 2) generate and optimize synthetic biology tools for in vivo circuit control that will be modular and widely applicable to probiotic engineering, and 3) explore the efficacy of an alternative, continual-delivery therapeutic for PKU. The proposed research is impactful because it will 1) develop reliable probiotic colonizers, 2) update the toolbox for synthetic biology in vivo applications, and 3) establish engineered probiotics as vehicles for sustained therapeutic delivery or controlled modulation of gut community architectures.